1. Field of Industrial Application
This invention relates to a stable immobilized alcohol oxidase, the method for preparing it and a highly accurate and stable alcohol measuring apparatus using it. The invention further relates to a highly accurate and stable alcohol measuring apparatus which can measure glucose at the same time.
2. Prior Art
Measuring apparatus using an immobilized enzyme are characterized in convenience, expediency and substrate specificity and are used in various fields such as clinical analysis, food analysis, and environmental instrumentation.
In the progress of the development of such a measuring apparatus using an immobilized enzyme, alcohol, particularly, ethanol analysis has been in particular demand in the fields of such as food, fermentation, and clinical field. However, with respect to the enzyme used for measurement of alcohol, none of sufficient stability has yet been obtained.
As an enzyme which can be used for alcohol measurement, there are known those such as alcohol dehydrogenase (EC.1.1.1. 1) and alcohol oxidase (EC.1. 1. 3. 13).
However, the alcohol dehydrogenase requires NAD (nicotinamide adenine dinucleotide) as a coenzyme, leading to higher cost of reagents used in analysis, and the stability of the enzyme is low. This enzyme is limited in used to measurement in a solution.
On the other hand, the alcohol oxidase is known to have been used in measurement after being immobilized.
However, such conventional immobilized alcohol oxidase is relatively low in activity and the stability is unsatisfactory in practical use.
U.S. Pat. No. 4,556,635, for example, describes a method for measuring alcohol concentration in water immiscible organic systems, but the preferable temperature is about 25.degree. C. accordingly, this enzyme is easily deactivated at relatively high temperature. Thus, using a conventional immobilization method, this measuring method has a substantial limitation in the practical use of alcohol oxidase.
U.S. Pat. No. 4,900,666 discloses a measuring method and test device using alcohol oxidase. However, as described in this specification, the alcohol oxidase is degenerated very readily, and various stabilizers have been studied, but those having sufficient effect for application to a measuring apparatus using immobilized enzyme have not yet been obtained.
In addition, these measuring apparatuses using conventional immobilized enzyme, have a defect, that is, after the high concentration alcohol exceeding the measuring range has been measured, response to the low concentration alcohol becomes smaller, that is, the sensitivity is varied.
In the field of fermentation and foods, the necessity of simultaneous measurement of glucose, a sweetener or raw material, and alcohol, a product or main component, is high. However, use of the conventional unstable immobilized alcohol oxidase could not afford development of a satisfactory simultaneous measuring apparatus.
Accordingly, it is an object of the present invention to provide a stable immobilized alcohol oxidase and a high-accuracy and stable alcohol measuring apparatus using it, particularly, to provide an alcohol measuring apparatus with excellent stability at room temperature or higher and of no sensitivity variation after measurement of the high concentration alcohol.
Another object of the invention is to provide a high-accuracy and stable alcohol measuring apparatus which can measure glucose at the same time.
These and other objects of the invention will be explicit from the following description of the invention.